The retina is a multi-layered nervous tissue where light energy is converted into nerve impulses. The outermost layer of the retina, closest to the front of the eye, is a layer of neurons that includes ganglion cells. Behind the ganglion cells is a layer of integrating neurons, and behind the integrating neurons is a layer of photoreceptor cells, called rods and cones. Photoreception in rods and cones begins with absorption of light by a pigment in the cells, the absorbed light causing a receptor potential.
Forming an intimate structural and functional relationship with the photoreceptor cells is the retinal pigment epithelium, a monolayer of specialized, cuboidal cells located immediately behind the retina. The retinal pigment epithelial (RPE) cells provide support for the photoreceptor cells and carry on important physiological functions, including solute transport, phagocytosis and digestion of discarded outer segments of membranes shed from photoreceptor cells, and drug detoxication.
The RPE cells rest on a specialized basement membrane, called the Bruch's membrane, a membrane 1 to 5 microns in thickness and composed of collagen, laminin and other molecules.
Underlying the RPE cells is the choriocapillaris of the choroid tissue. The choriocapillaris contains the vasculature to provide nutrients and remove metabolic by-products from the retina. Underlying the choroid tissue is the sclera.
It is believed that failure of the RPE cells to properly perform their functions alters the extracellular environment for photoreceptor cells, and leads to the eventual degeneration and loss of photoreceptor cells. Dysfunction of RPE contributes to the pathogenesis of a variety of sight-threatening diseases including age-related macular degeneration (ARMD) 1, serious retinal detachment 2, and such genetic diseases as gyrate atrophy 3 and choroideremia 4.
Age-Related Macular Degeneration
ARMD is the leading cause of visual impairment in western countries and is believed to be caused by progressive deterioration of RPE, Bruch's membrane, and the choriocapillaris, which leads to subsequent damage to the photoreceptor cells. In ARMD, the RPE cells are dysfunctional. In one form of ARMD, degeneration of the RPE cells is followed by atrophy of the choriocapillaris. In another form, the Bruch's membrane is altered and degraded by invasion of choroid neovascular membrane (CNV) into the subretinal space, leading to hemorrhage in the subretinal space, and scarring, with possible further damage to both RPE and photoreceptors.
Although CNV invasion 5 into the subepithelial and/or subretinal space can be treated with laser photocoagulation, if neovascularization is subfoveal, the results are poor 6. Surgical removal of CNV membranes seldom leads to improvement of vision or halts the progression of ARMD 7. The poor results may be due to inadvertent removal of RPE during the CNV removal 8, the failure of RPE to re-populate, the progressive enlargement of choriocapillaris atrophy following submacular surgery 9, and photoreceptor loss.
Problems with Prior Art Methods of RPE Transplantation
Only limited success in restoring vision using current methods of RPE transplantation has been achieved with either autologous or allogeneic sources (experimental 6;10 and clinical 11-14). In experimental animals, in particular in the Royal College of Surgeons ARCS) Rat model of retinal degeneration 15-19, RPE transplantation has been used to rescue photoreceptors, preserve choriocapillaris, and prevent CNV. In the case of allogeneic RPE transplantation, one obvious reason to explain the failure is allograft rejection 13;20. However, in the case of autologous RPE transplantation, the failure to restore vision may be due to the failure of transplanted RPE to repopulate the diseased site or to function in vivo. The failure of the RPE to grow or to function may be due to damage to the Bruch's membrane.
The Bruch's Membrane
There is evidence that the integrity of Bruch's membrane is crucial for RPE repopulation and subsequent functions. For example, surgical removal of RPE without damage to Bruch's membrane results in partial regeneration of the RPE monolayer in the non-human primate and domestic pig with the preservation of the underlying choriocapillaris and the overlying photoreceptors 21-23. In contrast, abrasive debridement causes more damage to Bruch's membrane, leads to incomplete repopulation of RPE, choriocapillaris atrophy, and outer segment retinal degeneration 24. Experimental transplantation of cultured human RPE to Bruch's membrane of the owl's monkey eye results in normal attachment, viability, and expressing junctions and morphological polarity 25. Autologous transplantation of RPE onto an abrasively debrided Bruch's membrane decreases choriocapillaris atrophy and photoreceptor loss in rabbits 26.
In the case of human patients with ARMD, the failure of restoring RPE function in transplanted human autologous RPE may be at least partially due to the altering of Bruch's membrane intrinsically caused by ARMD 27 and damaged by surgical removal of CNV membrane 23;24.
The current method of RPE transplantation, subretinal injection of an RPE cell suspension, achieves a limited success. There are many problems associated with this method, including a resulting subretinal fibrosis and the formation of multiple layers of RPE 6. These problems may be due to lack of restoration of in vivo (normal) epithelial phenotype and function. To date, no advance has been made in restoring Bruch's membrane in the surgical treatment of ARMD.
Immunological Aspects of RPE Transplantation
Although the eye as a part of the central nervous system has characteristics of an immunologically privileged site, it has been demonstrated that RPE transplants sensitized their recipients to both alloantigens and to RPE-specific autoantigens. Both are considered potential barriers to successful transplantation, and would make immune suppression regimens necessary 28. It was also demonstrated that the immunological response is most likely related to the amount of transplanted cells and that the response increases with time. RPE allografts in the RCS rat were not rejected for up to one year.
Problems with Prior Art Substrates and Methods of Culturing RPE
Substrates that have been used for this purpose include plastic 31, cross-linked collagen 32, gelatin 1, fibrinogen 2, poly-L-lactic acid ELLA) 3, PLLA/PLGA (poly-DL-lactic-co-glycolic acid) film 5-6, hydrogel 7, and basement membrane-containing anterior lens capsule 7. There are many disadvantages associated with each of the prior art substrates used for culturing RPE cells for transplantation, and a number of problems remain unsolved.
Impermeable Substance
One attempt at RPE transplantation utilized RPE cells isolated from either the whole eye or from a biopsy with Dispase® (GodoShusei Co., Ltd., Tokyo, Japan), and seeded on an impermeable substrate such as the plastic dish 31. These cells were prepared as a dissociated cell suspension 6;10 or as a patch derived from fetus 11 before transplantation. These cells did not fully retain their epithelial morphology. Furthermore, pigmentation of melanolipofuscin granules rapidly disappears on plastic cultures 33.
Porous Support (Cross-Linked Collagen, Collagen, Gelatin, Fibrinogen, PLLA/PLGA, Hydrogels, CNV Membranes, Lens Capsule)
Cross-linked collagen, when used for transplantation, is damaging to the retina due to its thickness, poor permeability and inability to degrade 32. Although human RPE cells 34 seeded on collagen membrane produced a monolayer of cells that exhibited a measurable transepithelial resistance and electrical potential 35, the cells did not achieve the in vivo state of development and function.
Gelatin has been used as an embedding medium, but not as a substrate for attachment. Fibrinogen and PLLA microspheres are also not suitable for transplanting RPE as a single sheet when transplanted to the subretinal space 2;3. PLLA/PLGA films do provide the RPE monolayer sheet for transplantation, but in vitro cultures of human fetal RPE cells grown on these supports do not show pigmentation (melanogenesis) 5;6. Hydrogel also provides the RPE monolayer sheet for transplantation, but the resultant cell density and the cell tight junction determined by expression of ZO-1, is relatively low 7.
Human RPE cells have also been cultured on surgically excised CNV membranes from ARMD patients, but the culture forms multiple layers 36.
Although lens capsule is a basement membrane-containing, natural material, it is not an ideal substrate for RPE culture and transplantation. Anterior lens capsule has been used to grow RPE 7;8 and IPE 8, and to transplant RPE and IPE 8 with lens capsule to the subretinal space. Both hydrogel and lens capsule, when used as substrates for RPE cultures, do not allow pigmentation to form (or melanogenesis) by RPE cells in culture 7;8.
The inventors of the present subject matter attempted to use the lens capsule as an autologous substrate for RPE/IPE transplants. However, the tendency of the capsule to curl made this technique impracticable. The idea to use the posterior capsule, because it is thinner, was also abandoned, for a number of reasons. First, the posterior capsule is difficult to obtain during surgery, without putting the patient at a high risk. Secondly, no absorption or slow absorption of the lens capsule material might inhibit the survival of the transplanted RPE cells because of insufficient contact of the cells with the Bruch's membrane and/or choriocapillaris.
Recently Bilbao et al, 37 disclosed the use of PLGA, coated on one side of a lens capsule to prevent curling and to facilitate its use for subretinal release. However, histological studies showed not only that the PLGA had completely dissolved after 4 weeks, but also that the overlying retinal layers were disrupted, the disruption accompanied by a large amount of cell infiltration.
Cryoprecipitate from blood donors was also tested as a possible autologous substrate for human fetal retinal pigment epithelium by Farrokh-Siar et al in 1999 (38). Dutt et al, 39 used several substrates for culture of BPE cell line 0041: extracts from placenta and amnion; MATRIGEL® (Collaborative Biomedical Products. Inc., Bedford, Mass.), a commercially available basement membrane matrix; dishes coated with extracellular matrix secreted by endothelial cells (ECM); dishes coated with collagen IV and/or laminin; dishes coated with collagen I and/or fibronectin. Although deeply pigmented, cells grown on MATRIGEL® looked like fibroblasts.
As described above, problems that remain to be solved include, for example, maintenance of the morphology of the RPE phenotype in cultured and transplanted RPE cells; creation of a uniform monolayer of autologous RPE on a biocompatible substrate; improvement of the transplant technique to better cover the defect; overcoming immune rejection of RPE transplants due to both alloantigens and RPE-specific auto-antigens; and prevention of subretinal fibrosis following RPE transplantation.
Amniotic membrane is a biological membrane that lines the inner surface of the amniotic cavity and comprises a simple, cuboidal epithelium, a thick basement membrane, and an avascular mesenchymal layer containing hyaluronic acid. Amniotic membrane transplantation has been used for ocular surface reconstruction in the treatment of acute chemical and thermal burns of corneal tissue 53.
Overall, the medical need for a method of culturing RPE cells suitable for transplantation to the subretinal space, a suitable RPE transplant composite with actions to maintain the epithelial phenotype and exert anti-inflammatory, anti-scarring, and anti-angiogenic effects to the underlying stroma, and a method of transplanting RPE cells to the subretinal space, has not been met.